Medical use of matrix metalloproteinase inhibitors for inhibiting tissue contraction

ABSTRACT

The use of an MMP inhibitor, especially a collagenase inhibitor, in the manufacture of a medicament for the treatment of a natural or artificial tissue comprising extracellular matrix components to inhibit contraction of the tissue and methods for the treatment of tissue comprising extracellular matrix components to inhibit contraction.

RELATEDNESS OF THE INVENTION

The subject invention is a U.S. national stage application ofPCT/GB95/00576, filed Mar. 16, 1995, claiming priority based on GB9405076.2 filed on Mar. 16, 1994, which are incorporated herein in theirentirety by reference.

The present invention relates to matrix metalloproteinase (MMP)inhibitors, especially collagenase inhibitors, and to their use in themanufacture of medicaments.

There are many different types of collagen found in the body and they,together with other extracellular matrix components, for example,elastin, gelatin, proteoglycan and fibronectin, make up a largeproportion of the body's extracellular tissue. Matrix metalloproteinases(MMPs) are enzymes that are involved in the degradation and denaturationof extracellular matrix components. Collagenases, for example, arematrix metalloproteinases that degrade or denature collagen.

A large number of different collagenases are known to exist. Theseinclude interstitial collagenases, type IV-specific collagenases andcollagenolytic proteinases. Collagenases are generally specific forcollagens which, in their full triple helix structure, are extremelyresistant to other enzymes.

Other MMPs are involved in the degradation and denaturing of differentextracellular matrix components, for example, elastin, gelatin andproteoglycan. Some MMPs are able to degrade or denature severaldifferent types of collagen and also other extracellular matrixcomponents. For example, stromelysin degrades type IV collagen, which isfound in basement membrane, and also has an effect on otherextracellular matrix components such as elastin, fibronectin andcartilage proteoglycans.

There is a classification system for MMPs, see Nagase et al 1992. Forexample, MMP1 is a collagenase that is sometimes called "collagenase",MMP2 is a 72 kD gelatinase, MMP3 is stromelysin and MMP9 is a 92 kDgelatinase. The official designations are used herein.

Collagenases have been implicated in a number of diseases, for example,rheumatoid arthritis [Mullins, D. E. et al 1983], periodontal diseaseand epidermolysis bullosa, and it has been proposed to use MMPinhibitors in the treatment of such conditions.

U.S. Pat. No. 5,183,900, No. 5,189,178 and No. 5,114,953 describe thesynthesis ofN-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophanmethylamide, also known as GM6001, Galardin or Galardin-MPI (tradenames), and other MMP inhibitors, and their use in the prevention andtreatment of corneal ulceration. Treatment of corneal ulcers withpeptide hydroxamic acid inhibitors has been found to assist in thehealing of those ulcers. Further details of such uses are given inSchultz et al 1992.

Also described in the above-mentioned US patent specifications is theuse of collagenase inhibitors in situations where bacterial enzymes maybe detrimental to tissue, for example, in bacterial ulceration.

Other collagenase inhibitors based on hydroxamic acid are disclosed inWO 90/05716, WO 90/05719 and WO 92/13831. Such collagenase inhibitorsare disclosed as being used in the management of disease involvingtissue degradation, particularly disease involving collagen breakdown,and/or the promotion of wound healing.

Other synthetic MMP inhibitors and, in particular, collagenaseinhibitors that have been developed include those described inEP-A-126,974 and EP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and4,743,587.

One inhibitor undergoing clinical trials is BB-94 also known asBatimastat (British Bio-technology Ltd.). Potential uses of BB-94 forthe control of cancer metatasis are described in EP-A-276436. It hasbeen proposed to use an oral formulation of BB-94 in the treatment ofbone cancer.

The present invention is concerned with the contraction of tissues, forexample, scars. Contraction of tissues comprising extracellular matrixcomponents, especially of collagen-comprising tissues, may occur inconnection with many different pathological conditions and with surgicalor cosmetic procedures. Contracture, for example, of scars, may causephysical problems, which may lead to the need for medical treatment, orit may cause problems of a purely cosmetic nature.

It has been proposed that contraction is cell-mediated and a number ofstudies have suggested possible mechanisms for cell mediated collagencontraction [Gabbiani et al. 1972, Ehrlich & Rajaratnam 1990].Investigations have been made into the role, if any, played by MMPs inthe process of contracture. However, according to one proposition, MMPsare not produced during contraction [Schor et al. 1980]. According toanother proposition, MMPs are produced during lattice contraction butare not implicated in the contractile process [Nakagawa et al. 1989,Mauch et al. 1989, Lambert 1992]. Instead, it has been proposed thatcontraction is dependent upon the extracellular lattice cell number,upon there being an intact actin cytoskeleton, and upon attachment ofthe cells to the extracellular matrix.

The present invention is based on the surprising observation that,during experiments on in vitro models of scar contraction, collagen (themain component of scar tissue) appears to be invaded and permanentlyremodelled by fibroblasts and that such invasion and remodelling isinhibited by collagenase inhibitors. The remodelling generally appearsas contraction of the collagen, which contraction is inhibited byinhibition of collagenase. Furthermore, inhibition of other MMPs alsoresults in inhibition of contraction. The observation that contractionof the tissue involves MMPs is particularly surprising since previousinvestigations have shown that MMPs are not produced during contractionwhile other investigations have indicated that MMPs are produced but arenot involved in the contractile process (see above).

The present invention provides the use of an MMP inhibitor in themanufacture of a medicament for the treatment or prophylaxis of anatural or artificial tissue comprising extracellular matrix componentsto inhibit, i.e. restrict, hinder or prevent, contraction of the tissue,especially contraction resulting from a pathological condition or fromsurgical or cosmetic treatment.

The present invention also provides a method for the inhibition in vivoor in vitro of contraction of a natural or artificial tissue comprisingextracellular matrix components, which comprises administering an MMPinhibitor to the tissue during and/or after its formation. Under in vivoconditions, a therapeutically effective amount of the MMP inhibitorshould be administered.

The present invention especially provides the use of a collagenaseinhibitor in the manufacture of a medicament for the treatment orprophylaxis of tissue comprising collagen to inhibit contraction of thetissue resulting from contraction of the collagen.

Further, present invention especially provides a method for theinhibition of contraction of tissue comprising collagen, resulting fromcontraction of the collagen, which comprises administering atherapeutically effective amount of a collagenase inhibitor to thetissue.

The methods of the present invention may be used for medical or cosmetictreatment.

The invention provides a method for the inhibition, for cosmeticreasons, of disfigurement caused by contraction of tissue comprisingextracellular matrix components, which method comprises administering amatrix metalloproteinase inhibitor to the tissue.

Cosmetic treatments, such as chemical or physical dermal abrasion, usedas anti-ageing treatments, cause trauma to the skin. Use of MMPinhibitors during the healing process which occurs after the initialabrasion is a cosmetic use of MMP inhibitors according to the presentinvention.

The present invention also provides the use of an MMP inhibitor toinhibit, i.e. restrict, hinder or prevent, invasion by cells, especiallyfibroblasts, into tissue comprising an extracellular matrix and/ormigration by cells, especially fibroblasts, in or through tissuecomprising an extracellular matrix.

The term "MMP inhibitor" is used herein to denote any substance that iscapable of inhibiting, i.e. restricting, hindering or preventing, theaction of an MMP. The term "collagenase inhibitor" is used herein todenote any substance that is capable of inhibiting, i.e. restricting,hindering or preventing, the action of a collagenase. A collagenaseinhibitor may be specific for one particular collagenase or may inhibitseveral different collagenases. A collagenase inhibitor may also inhibitother MMPs, in which case it may also be defined as an MMP inhibitor.

The term "inhibitor" as used herein includes agents that act indirectlyby inhibiting the production of the relevant enzyme, for example anantisense molecule, as well as agents that act directly by inhibitingthe enzyme activity of the relevant enzyme, such as, for example, aconventional inhibitor.

An MMP, e.g. collagenase, inhibitor may be naturally-occurring orsynthetic. An MMP, e.g. collagenase, inhibitor may be an anti-MMP, e.g.anti-collagenase, antibody, either polyclonal or monoclonal. Theinhibitory activity of a putative MMP inhibitor may be assessed by anymethod suitable for determining inhibitory activity of a compound withrespect to an enzyme. Such methods are described in standard textbooksof biochemistry. A more detailed description of MMP, e.g. collagenase,inhibitors is given below.

Collagen is the major component of scar and other contracted tissue andas such is the most important structural component to consider.Nevertheless, scar and other contracted tissue also comprises otherstructural components, especially other extracellular matrix components,for example, elastin, which may also contribute to contraction of thetissue. MMPs are involved in the synthesis and degradation of suchcomponents. In general, a collagenase inhibitor is used as the MMPinhibitor in accordance with the present invention but, it may beappropriate to use instead an inhibitor of an MMP other than acollagenase. It may be particularly advantageous to use a combination ofa collagenase inhibitor and one or more inhibitors of other MMPs or touse an inhibitor which inhibits both a collagenase and at least one ormore other MMPs.

The mechanism and control of contraction of tissues comprisingextracellular matrix components, for example, collagen-comprisingtissues, is still poorly understood. Some degree of contraction appearsto be part of the healing process, but the trigger for contraction isnot known. The involvement of MMPs in contraction of tissues, forexample, scar tissue, and the utility of MMP inhibitors according to thepresent invention in the inhibition, i.e. prevention, restriction andhindering, of contraction has been confirmed by the followingexperimental data:

When fibroblasts in an in vitro collagen gel model of scar contractionare subjected to antiproliferative agents after contraction hasoccurred, there is no significant expansion of the collagen gel, that isto say, no relaxation of the contraction, even when the fibroblast cellshave been killed and the supporting cytoskeleton of the cells removed.The remodelling of collagen leading to contraction therefore appears toresult from activity of one or more enzymic systems of the fibroblasts.

Fibroblasts involved in the contraction of collagen produce greateramounts of matrix metalloproteinase mRNAs and proteins than do controlfibroblasts. This is associated with cellular invasion of the collagen.Invasion of the collagen and contraction are inhibited by the use ofinhibitors specific to those MMPs coded for by the mRNAs which arepresent at higher levels in cells involved in contraction than incontrol cells:

Quantitative competitive reverse transcriptase polymerase chain reaction(QCRT-PCR) technique [Tarnuzzer & Schultz 1994] was used to study thelevels of MMP mRNA produced by human ocular fibroblasts in collagen TypeI lattices undergoing contraction in comparison with human ocularfibroblasts in monolayer cultures. It was found that in the collagenlattices the fibroblasts produced more mRNA for collagenase (MMP1), 72kD gelatinase (MMP2) and stromelysin (MMP3) but not for 92 kD gelatinase(MMP9) than did the control cells in the monolayer culture. Levels ofmRNA for MMPs 1, 2 and 3 in the lattices were found to be greater thanthose in the monolayer cultures; over 100 times greater at certain timesduring the contraction process (see Example 4 below). Gelatin zymography[Heussen & Dowdle 1980] was used to analyse and compare production ofthe gelatinolytic components of the cells. It was found thatgelatinolytic activity was increased compared to controls. It appeared,therefore, that the increased mRNA production resulted in increasedprotein production.

Antibodies against MMPs 1, 2, 3 and 9 were tested as contractioninhibitors. It was found that the antibodies against MMPs 1, 2 and 3gave significant inhibition of the contraction of collagen gels by humanocular fibroblasts but that the antibody against MMP9 did not result ininhibition of contraction.

In the present specification the term "contraction of collagen" includesnot only shrinkage of collagen but also any remodelling of collagen thatleads to contraction of the tissue comprising that collagen. It alsoincludes contributions made by other components of the tissue,especially other extracellular matrix components, for example, elastin.

As indicated above, contraction of tissues comprising extracellularmatrix components, especially of collagen-comprising tissues, may occurin connection with many different pathological conditions and withsurgical or cosmetic procedures. Contracture may cause physicalproblems, which may lead to the need for medical treatment, or it maycause problems of a purely cosmetic nature. It is therefore veryvaluable to have medicaments capable of inhibiting, i.e. restricting,hindering or preventing, such contraction. Important uses of suchmedicaments are described below. It should be understood, however, thatthe uses according to the present invention are not restricted to themanufacture of medicaments, or to methods of treatment, medical orcosmetic, suitable for the conditions described below. The presentinvention also includes use in the manufacture of medicaments or inmethods of treatment suitable for use in any case where contraction oftissue comprising extracellular matrix components resultingsubstantially from extracellular matrix component contraction isoccurring or may occur.

Although the discussion below refers specifically to collagencontraction and the use of collagenase inhibitors, broad spectrum MMPinhibitors and/or inhibitors of MMPs other than collagenases may be usedin inhibiting contraction of tissue comprising extra-cellular matrixcomponents and the present invention is to be understood as includingthe use of such MMP inhibitors as an alternative to the use ofcollagenase-specific inhibitors in the treatment of tissue comprisingextracellular matrix components. The present invention also includes theuse of broad spectrum MMP inhibitors and/or inhibitors of MMPs otherthan collagenases in addition to the use of collagenase inhibitors, forexample, in the treatment of tissue comprising extracellular componentsespecially collagen comprising-tissue.

Contraction of collagen-comprising tissue, which may also comprise otherextracellular matrix components, frequently occurs in the healing ofburns. The burns may be chemical, thermal or radiation burns and may beof the eye, the surface of the skin or the skin and the underlyingtissues. It may also be the case that there are burns on internaltissues, for example, caused by radiation treatment. Contraction ofburnt tissues is often a problem and may lead to physical and/orcosmetic problems, for example, loss of movement and/or disfigurement.The present invention therefore includes the use of MMP inhibitors, forexample, collagenase inhibitors, for example, in the form of amedicament, to inhibit contraction of the burnt tissue as it heals.

A further aspect of the present invention is the inhibition of thecontraction of skin grafts. Skin grafts may be applied for a variety ofreasons and may often undergo contraction after application. As with thehealing of burnt tissues the contraction may lead to both physical andcosmetic problems. It is a particularly serious problem where many skingrafts are needed as, for example, in a serious burns case.

An associated area in which the medicaments and methods of the presentinvention are of great use is in the production of artificial skin. Tomake a true artificial skin it is necessary to have an epidermis made ofepithelial cells (keratinocytes) and a dermis made of collagen populatedwith fibroblasts. It is important to have both types of cells becausethey signal and stimulate each other using growth factors. A majorproblem up until now has been that the collagen component of theartificial skin often contracts to less than one tenth of its originalarea when populated by fibroblasts. MMP inhibitors, for example,collagenase inhibitors may be used to inhibit the contraction to such anextent that the artificial skin can be maintained at a practical size.

One area of particular interest is the use of MMP, e.g. collagenaseinhibitors to prevent or reduce contracture of scar tissue resultingfrom eye surgery. Glaucoma surgery to create new drainage channels oftenfails due to scarring and contraction of tissues. A method of preventingcontraction of scar tissue formed in the eye, such as the application ofa suitable agent, is therefore invaluable. Such an agent may also beused in the control of the contraction of scar tissue formed aftercorneal trauma or corneal surgery, for example laser or surgicaltreatment for myopia or refractive error in which contraction of tissuesmay lead to inaccurate results. It is also useful in cases where scartissue is formed on/in the vitreous humor or the retina, for example,that which eventually causes blindness in some diabetics and that whichis formed after detachment surgery, called proliferativevitreoretinopathy. Other uses include where scar tissue is formed in theorbit or on eye and eyelid muscles after squint, orbital or eyelidsurgery, or thyroid eye disease and where scarring of the conjunctivaoccurs as may happen after glaucoma surgery or in cicatricial disease,inflammatory disease, for example, pemphigoid, or infective disease, forexample, trachoma. A further eye problem associated with the contractionof collagen-comprising tissues for which the methods and medicaments ofthe present invention may be used is the opacification and contractureof the lens capsule after cataract extraction.

Cicatricial contraction, contraction due to shrinkage of the fibroustissue of a scar, is common. In some cases the scar may become a viciouscicatrix, a scar in which the contraction causes serious deformity. Apatient's stomach may be effectively separated into two separatechambers in an hour-glass contracture by the contraction of scar tissueformed when a stomach ulcer heals. Obstruction of passages and ducts,cicatricial stenosis, may occur due to the contraction of scar tissue.Contraction of blood vessels may be due to primary obstruction orsurgical trauma, for example, after surgery or angioplasty. Stenosis ofother hollow visci, for examples, ureters, may also occur. Problems mayoccur where any form of scarring takes place, whether resulting fromaccidental wounds or from surgery. Medicaments comprising MMPinhibitors, e.g. collagenase inhibitors, may be used wherever scartissue is likely to be formed, is being formed or has been formed.

Conditions of the skin and tendons which involve contraction ofcollagen-comprising tissues include post-trauma conditions resultingfrom surgery or accidents, for example, hand or foot tendon injuries,post-graft conditions and pathological conditions, such as scleroderma,Dupuytren's contracture and epidermolysis bullosa. Scarring andcontraction of tissues in the eye may occur in various conditions, forexample, the sequelae of retinal detachment or diabetic eye disease (asmentioned above). Contraction of the sockets found in the skull for theeyeballs and associated structures, including extra ocular muscles andeyelids, may occur if there is trauma or inflammatory damage. Thetissues contract within the sockets causing a variety of problemsincluding double vision and an unsightly appearance.

Although the above discussion relates in particular to humans, animalsmay exhibit the conditions described above or similar or analogousconditions. The present invention therefore also relates analogously tomedicaments and methods for use in veterinary practice for the treatmentand care of animals and especialy for use in the treatment and care ofmammals.

The present invention provides a method of treating a human or othermammal to inhibit contraction of tissue comprising an extracellularmatrix component, especially contraction associated with a chemicalburn, a thermal burn or a radiation burn, a skin graft, a post-traumacondition resulting from surgery or an accident, glaucoma surgery,diabetes associated eye disease, scleroderma, Dupytren's contracture,epidermolysis bullosa or a hand or foot tendon injury, which comprisesadministering to the human or other mammal a therapeutically effectiveamount of an MMP inhibitor.

It appears that MMP inhibitors, e.g. collagenase inhibitors, inhibitcontraction tissues comprising extracellular components, for example,collagen, caused by cells such as fibroblasts but do not appear to beable to bring about significant reversal of such contraction.Accordingly, tissue which is being affected should generally be treatedat the time when the contraction is occurring. Preferably treatmentshould take place as early as possible, advantageously as soon as, andmost advantageously before, the first signs of contraction are observed.In treatments, conditions or healing processes where contraction ofextracellular component, e.g. collagen, comprising tissue is common. MMPinhibitors, e.g. collagenase inhibitors, may be used as a routineprophylactic measure before any signs of contraction have actually beenseen.

Since active contraction appears to be associated with active productionof MMPs, the treatment used to prevent the contraction should becontinued over at least the period during which contraction is likely tooccur. This may often be quite a significant period of time, forexample, several years or even longer. Contraction may still occur evenafter an initially open wound appears to have healed, for example, inpatients with burns. Also conditions such as hand tendon contractioninvolve contraction even though there is no wound as such.

As indicated above, the present invention involves the use of MMPinhibitors, especially collagenase inhibitors.

Both natural and synthetic MMP inhibitors (inhibitors of enzymeactivity), including collagenase inhibitors, are known.Naturally-occurring MMP inhibitors include α₂ -macroglobulin, which isthe major collagenase inhibitor found in human blood [Eisen et al 1970].Naturally occurring MMP inhibitors are also found in tissues. Thepresence of tissue inhibitors of MMPs has been observed in a variety ofexplants and in monolayer cultures of mammalian connective tissue cells[Vater et el 1979 and Stricklin and Wegus 1983]. Not only collagenaseinhibitors but also inhibitors for other MMPs, for example, gelatinaseand proteoglycanase are found. MMP inhibitors are generally unable tobind the inactive (zymogen) forms of the respective enzymes but complexreadily with active forms [Murphy et al 1981]. Tissue MMP inhibitors arefound, for example, in dermal fibroblasts, human lung, gingival, tendonand corneal fibroblasts, human osteoblasts, uterine smooth muscle cells,alveolar macrophages, amniotic fluid, plasma, serum and the α-granule ofhuman platelets [Stricklin and Wegus 1983; Welgus et al 1985; Welgus andStricklin 1983; Bar-Sharvit et al 1985; Wooley et al; 1976; and Cooperet al 1985].

Synthetic collagenase inhibitors and inhibitors for other MMPs have beenand are being developed. Compounds such as EDTA, cysteine, tetracyclineand ascorbate are all inhibitors of collagenases but are relativelynon-specific. As indicated above, synthetic inhibitors that have definedspecificity for MMPs, including collagenase inhibitors, are described inthe literature. For example, U.S. Pat. Nos. 5,183,900, 5,189,178 and5,114,953 describe the synthesis ofN-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophanmethylamide, also known as GM6001 or Galardin (trade name), and otherMMP inhibitors. Other collagenase inhibitors based on hydroxamic acidare disclosed in WO 90/05716, WO 90/05719 and WO 92/13831. Furthersynthetic MMP inhibitors and in particular collagenase inhibitors thathave been developed include those described in EP-A-126,974 andEP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and 4,743,587. Yet anotherinhibitor is BB-94, also known as Batimastat (British Bio-technologyLtd.), see for example, EP-A-276436. Disclosed in WO90/05719 as havingparticularly strong collagenase inhibiting properties are[4-(N-hydroxyamino)-2R-isobutyl-3S-(thio-phenyl-thiomethyl)succinyl]-L-phenylalanine-N-methylamide(especially good) and[4-(N-hydroxyamino)-2R-isobutyl-3S-(thiomethyl)succinyl]-L-phenylalanine-N-methylamideand in WO90/05716[4-(N-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-(3-aminomethylpyridine)amide and[4-N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylalanine-N-[4-(2-aminoethyl)-morpholino]amide.

The contents of the patent specifications and literature referencesmentioned herein are hereby incorporated by reference.

As indicated above, the properties of natural and synthetic collageninhibitors may vary. Individual inhibitors often have differentspecificities and potencies. Some inhibitors are reversible, others areirreversible. In general the more potent an inhibitor's inhibitoryeffects on a collagenase the better. For some uses an inhibitor specificto one particular collagenase may be required but generally a broadspectrum MMP inhibitor, for example, GM6001 (Galardin (trade name)), ispreferred.

GM6001 (Galardin (trade name)) is a very potent MMP inhibitor that iseffective against collagenase. It has the structure: ##STR1##

A detailed account of its ability to inhibit human skin fibroblastcollagenase, thermolysin and Pseudomonas aeruginosa elastase is given inGrobelny et al, 1992. Its inhibition constants with three types of MMPsare now calculated to be:

    ______________________________________                                        collagenase         Ki = 0.4nmol/l                                            gelatinase          Ki = 0.4nmol/l                                            stromelysin         Ki = 20nmol/l                                             ______________________________________                                    

Preferred MMP inhibitors for use according to the present inventioninclude GM6001 (Galardin) and those synthetic inhibitors described andreferred to above. Preferred inhibitors include peptide hydroxamic acidsor pharmaceutically acceptable derivatives thereof. Especially preferredare those compounds that are described and claimed in U.S. Pat. No.5,189,178; No. 5,183,900 or No. 5,114,953 and that are collagenaseinhibitors. Those with low Ki values, i.e. high pKi values are generallypreferred. GM6001 (Galardin (trade name)) is an MMP inhibitor that isespecially preferred because it is one of the most potent collagenaseinhibitors known at present. However, for certain applications it may bepreferable to use a less potent (weaker) inhibitor.

The preferred broad spectrum MMP inhibitor for use in accordance withthe present invention is GM6001 (Galardin (trade name)). It is able toinhibit the action not only of collagenases but of other MMPs as well.

Also preferred are inhibitors that are capable of inhibiting MMPs 1, 2and 3 (collagenase, 72kD gelatinase and stromelysin, respectively).These may be used individually or in combination.

As indicated above, an anti-MMP polyclonal or monoclonal antibody,especially an anti-collagenase antibody, may be used as an inhibitor. AnMMP antigen may be used in immunisation protocols to obtain polyclonalantisera immunospecific for that enzyme. The antigen may be a haptenderived from an MMP, especially from an active site region, or may be afull-length MMP or a fragment thereof. Using standard protocols andmammalian subjects, such as rabbits or mice, polyclonal antibodies maybe obtained. Those may then be used as inhibitors. Monoclonal antibodiesmay be produced according to standard procedures, for example, using anappropriate MMP antigen, for example, a collagenase antigen.

Antibodies which are specific for a particular MMP may be made and theuse of such specific inhibitors may be preferred under certaincircumstances. For example, an antibody to MMP1, MMP2 or MMP3(collagenase, 72kD gelatinase or stromelysin respectively) or a mixtureof two or more thereof may be used.

An alternative method of inhibiting the action of an MMP is to reducethe amount of the MMP by preventing its production. One method ofpreventing protein production is by the use of antisense nucleic acidmolecules. An antisense molecule need not be large; 20 base pairs isoften sufficient. If the molecule is small it should be able to enterthe cells unaided but liposomes can be used to assist entry if required.An antisense molecule will usually be designed to attach to the MMP mRNAbut may be designed to attach to the appropriate DNA during replicationand transcription.

Although antibodies usually bind to proteins it is possible to produceantibodies which bind to nucleic acids. Accordingly, there may be usedas an inhibitor according to the present invention an antibody thatbinds to the mRNA or the DNA of the selected MMP and hence hindersproduction of the MMP.

Reducing the production of an MMP has the advantage that it should bepossible to use a smaller amount of inhibitor than is required fordirect inhibition of MMP enzyme activity because each MMP mRNA moleculeand the MMP DNA is responsible for the production of many MMP enzymemolecules.

Inhibitors for use according to the present invention must be able to beused in high enough concentrations and large enough doses to giveadequate inhibition without being toxic to cells with which they comeinto contact.

For the treatment of some conditions it may be preferred to use amedicament containing a collagenase inhibitor and at least one otherenzyme inhibitor. That additional inhibitor may also have collagenaseinhibitory properties and/or it may have inhibitory properties for adifferent enzyme, for example, for a different MMP. If two or moreinhibitors are used then the second and any additional inhibitorpreferably has inhibitory properties for an enzyme other than acollagenase. Additional inhibitors may be, for example, inhibitors ofother MMPs such as a gelatinase or a stromelysin, or inhibitors of otherenzymes that break down tissue such as serine proteases, for example, aserine protease inhibitor such as aprotinin may be used.

Cytokines, for example, interleukin-1, in the environment ofcollagen-comprising tissue may stimulate collagenase production and soit may also be preferable to include an inhibitor of cytokines in themanufacture of medicaments or in methods of treatment according to thepresent invention.

Medicaments according to the present invention are generally provided ina pharmaceutical preparation form suitable for topical administration,for example, an emulsion, suspension, cream, lotion, ointment, drops,foam or gel. Such preparations are generally conventional formulations,for example, as described in standard text books of pharmaceutics suchas the British Pharmacopoeia. Other suitable pharmaceutical forms fortopical administration include dry powders, aerosols and sprays, whichmay be especially suitable for application to burns. Further suitablepharmaceutical preparation forms include those for administration byinjection or infusion, for example, sterile parenteral solutions orsuspensions, especially for administration directly into, or into thearea of, the extracellular matrix component, e.g. collagen, comprisingtissue, for example, by subconjunctival, subcutaneous, interpleural orintra-peritoneal injection, and also slow release delivery systems, forexample, liposome systems.

The invention especially provides a pharmaceutical preparation (otherthan a preparation suitable for use in the eye) suitable for applicationto a wound, including an ulcer, a burn or skin graft comprising a matrixmetalloproteinase inhibitor. Advantageously, the preparation is in theform of a solution, suspension, cream, ointment, or gel, in which theMMP inhibitor is in a concentration of from 0.4 μg/ml to 400 μg/ml. TheMMP inhibitor is preferably a collagenase inhibitor.

Oral formulations may also be used. These may be in the form of tablets,capsules, powders, granules, lozenges or liquid or gel peparations.Tablets may be coated by methods well known in normal pharmaceuticalpractice. Liquid formulations include syrups. Oral formulations may beused to treat directly conditions such as stomach ulcers and may also beused to treat conditions systemically.

The inhibitor(s) may be dissolved or dispersed in a diluent or carrier.The choice of carrier depends on the nature of the inhibitor, itssolubility and other physical properties, and on the method and site ofapplication. For example, only certain carriers are suitable forpreparations for use in the eye.

Carriers include ethylene glycol, silver sulphadiazine cream andhypromellose. These may be used in creams and drops. An acetate buffersystem may also be used. Further pharmaceutically suitable materialsthat may be incorporated in pharmaceutical preparations includeabsorption enhancers, pH regulators and buffers, osmolarity adjusters,emollients, dispersing agents, wetting agents, surfactants, thickeners,opacifiers, preservatives, stabilizers and antioxidants, foaming agentsand flocculants, lubricants, colourants and fragrances (generally onlyin primarily cosmetic preparations).

Gels and liposomes may be the preferred delivery method when theinhibitor is an antisense molecule.

Preferably a medicament according to the present invention is applieddirectly to an open wound or is injected directly into the site oftissue contraction. Suitable medicaments may, however, be applied to theskin surface where the tissue to be treated is below that surface, theactive ingredient then being absorbed by and passing through the skin.Penetration enhancers are preferably incorporated in such medicaments.

Medicaments according to the present invention comprising MMPinhibitors, for example, collagenase inhibitors, for use in theinhibition of the contraction of tissues comprising extracellular matrixcomponents, for example, collagen-comprising tissues, may containfurther pharmaceutically active ingredients, for example antibiotics,antifungals, steroids, and further enzyme inhibitors, for example,serine protease inhibitors (as described above). Further components forcertain indications include growth or healing promoters such asepidermal growth factor (EGF), fibronectin and aprotinin. As mentionedabove cytokine inhibitors may also be included.

The inhibitors will generally be used in liquid and other non-solidformulations having concentrations of around 0.4 to 400 μg/ml. In somecases, however, higher concentrations may be required. The total amountused and the dose administered will depend on the severity and area ofthe contraction, the condition causing it and the physicalcharacteristics of the patient and the site and method ofadministration.

The following non-limiting Examples illustrate the invention. TheExamples relate to experiments carried out in vitro; the teachings aredirectly applicable in vivo, for example, to the treatment of humans andmammals, for example, as described in the specification.

The Examples illustrate the use of MMP inhibitors in in vitro models ofscar contraction and of artificial skin. They also include experimentswhich illustrate the lack of toxicity to cells of MMP inhibitors andtheir effect on cell morphology. Further experiments investigate thelevels of some MMP mRNA and of some MMPs present in cells duringcontraction.

FIG. 1 is a diagrammatic representation of the results of theexperiments described in Example 1. It is a graph showing the area offibroblast populated collagen gel over time for a number of differentculture regimes.

FIG. 2 is a diagrammatic representation of the results of theexperiments described in Example 2. It is a histogram showing counts perminute of radiation emitted by cells grown under a number of differentconditions.

FIG. 3A is a diagrammatic representation of the results of experimentsdescribed in Example 3(a). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 3B is a diagrammatic representation of the results of experimentsdescribed in Example 3(a). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 3C is a diagrammatic representation of the results of experimentsdescribed in Example 3(b). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 3D is a diagrammatic representation of the results of experimentsdescribed in Example 3(c). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 3E is a diagrammatic representation of the results of experimentsdescribed in Example 3(c). It is a graph showing the mean number ofcells per well against time under a number of different culture regimes.

FIG. 3F is a diagrammatic representation of the results of experimentsdescribed in Example 3(c). It is a graph showing the percentage ofviable cells against time under a number of different culture regimes.

FIG. 3G is a diagrammatic representation of the results of experimentsdescribed in Example 3(d). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 3H is a diagrammatic representation of the results of experimentsdescribed in Example 3(c). It is a graph showing mean lattice area offibroblast populated collagen gels over time for different cultureregimes.

FIG. 4A is a diagrammatic representation of the results of theexperiments described in Example 5(i). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

FIG. 4B is a diagrammatic representation of the results of theexperiments described in Example 5(ii). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

FIG. 4C is a diagrammatic representation of the results of theexperiments described in Example 5(iii). It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

FIG. 5 is a diagrammatic representation of the results of theexperiments described in Example 6. It is a graph showing the meanlattice area of fibroblast populated collagen gels over time under twodifferent culture regimes.

FIG. 6 is a reproduction of a photograph showing the results of the PCRreactions described in Example 4.

FIG. 7 is a reproduction of a photograph showing the results of thegelatin zymography analyses described in Example 4.

FIG. 8A and FIG. 8B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing the degree of contractionas described in Example 8. FIG. 8A shows a control gel and FIG. 8B showsa gel exposed to inhibitor.

FIG. 9A and FIG. 9B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing the development of actinstress fibres on the surface of the cells as described in Example 8.FIG. 9A shows a control gel and FIG. 9B shows a gel exposed toinhibitor.

FIG. 10A and FIG. 10B are each a reproduction of a photograph offibroblasts grown in monolayers showing the presence of stress fibres asdescribed in Example 8. FIG. 10A shows a control gel and FIG. 10B showsa monolayer exposed to inhibitor.

FIG. 11A and FIG. 11B are each a reproduction of a photograph of acollagen gel seeded with fibroblasts showing cellular attachments asdescribed in Example 8. FIG. 11A shows a control gel and FIG. 11B showsa gel exposed to inhibitor.

EXAMPLES Example 1

Preparation of Materials

(a) Collagen Solutions

Solutions of type 1 collagen were prepared by dissolving 100 mg ofcollagen in 20 ml of 0.1% (v/v) glacial acetic acid in distilled water.The collagen was sigma type 1 collagen and was prepared by the method ofBornstein MB Lab Invest 7134 1958.

(b) Concentrated Culture Medium

A concentrated tissue culture medium was prepared by mixing thefollowing:

35 ml distilled water

15 ml 10× MEM (Eagle's minimal essential medium)

1.5 ml glutamine

1.5 ml fungizone (amphotericin B 250 μg/ml of water)

1.5 ml 10,000 units penicillin/10 mg streptomycin per ml solution(solvent is water)

4 ml 7.5% (w/v) sodium bicarbonate solution

Solutions of 4.9 ml of the concentrated culture medium in 180 μl of 0.1Msodium hydroxide solution were used in the preparation of collagen gels.

(c) Collagenase Inhibitor Solutions and Control Solutions

Solutions having three different concentrations of collagenase inhibitor(400, 40 and 4 μg/ml), a buffer solution (control 1) and a normal growthmedium solution (control 2) were prepared as described below. Thecollagenase inhibitor used was GM6001 (Galardin (trade name)).

50 μl of glacial acetic acid was added to 11 mg of collagenase inhibitorand the inhibitor was allowed to dissolve. The pH of a 24.875 ml aliquotof serum-free HEPES (N-[2-hydroxyethylpiperazine-N'-[2-ethane sulfonicacid]) buffered DMEM (Dulbecco's Modified Eagle's Medium) was adjustedto pH 8 using sterile 1M sodium hydroxide solution and the aliquot wasthen added to the inhibitor solution. The resulting solution had aninhibitor concentration of 400 μg/ml.

The 400 μg/ml solution was serially diluted with serum-free HEPES-DMEMto give solutions containing the collagenase inhibitor at concentrationsof 40 and 4 μg/ml.

The solutions of all three concentrations were then supplemented with10% (v/v) newborn calf serum.

The buffer solution (control 1) was prepared by adding 50 μl of glacialacetic acid to a 24.875 ml aliquot of serum-free HEPES buffered DMEM ofpH 8 (pH adjusted with sterile 1M sodium hydroxide solution as above),then readjusting the pH to pH 7.4 with 1M sterile sodium hydroxidesolution and finally supplementing with 10% (v/v) of newborn calf serum(NCS).

The normal growth medium solution (control 2) was prepared bysupplementing an aliquot of serum-free HEPES buffered DMEM with 10%(v/v) newborn calf serum.

The pH and osmolarity of the test and control solutions were measured,see Table 1, Treatment of Gels with Test Solutions.

(d) Cell Cultures

Cultures of human ocular fibroblasts were grown, in the normal way (seeKhaw P. T. et al 1992 for the method of growth), until the monolayers(single layers of cells on a plastic culture disc, not embedded in amatrix) were just subconfluent. They were then removed from theirsubstratum via a trypsinisation and were pelleted by centrifugation at3000×g for 8 minutes. The supernatant was then discarded and the cellpellet resuspended in 1.1 ml of newborn calf serum. 100 μl of thesuspension was removed and counted in a Coulter counter (Model ZF).

(e) Collagen Gels

Each collagen gel had a final volume of 1.1 ml made up of 0.6 ml of asolution of type 1 collagen obtained according to (a) above, 0.35 ml ofconcentrated culture medium prepared according to (b) above, and 0.15 mlof cell suspension containing 100,000 cells prepared as described in (d)above.

Triplicate gels were made by adding 1.05 ml of the concentrated mediumto 1.8 ml of the type 1 collagen solution, mixing rapidly and thenadding 0.45 ml of a cell suspension containing 300,000 cells and mixingrapidly.

1 ml aliquots of this gel were then added to petri dishes (area=8 cm²)and then the dishes were rotated so that the gels were evenlydistributed across the bottoms of the dishes. The gels were thenincubated at 37° C. in 5% CO₂ in air until they had solidified (usually3 to 5 minutes). An area of the gel was then detached from the edge ofeach petri dish and 3 ml of normal growth medium solution (control 2),inhibitor test solution or buffer solution (control 1), were added. Eachgel was then fully detached from the edges and bottom of the petri dishso that free-floating gels were obtained.

Treatment of Gels with Test Solutions

The gels, after solidification, were treated for 24 hours with 3 ml ofeach of the following test solutions:

                  TABLE 1                                                         ______________________________________                                        Test Solution         pH    Osmolarity                                        ______________________________________                                        1) Collagenase inhibitor at 400μg/ml                                                             7.8   421                                               2) Collagenase inhibitor at 40μg/ml                                                              7.8   339                                               3) Collagenase inhibitor at 4μg/ml                                                               7.9   329                                               4) Buffer (control 1) 7.8   404                                               5) Normal growth medium (control 2)                                                                 7.8   331                                               ______________________________________                                    

The test and control solutions were replaced by 3 ml of the appropriatefresh solution after 24 hours. Photographs of the gel areas were takenon days 1 and 2 and were then digitised. The resulting data wereprocessed by a computer and the gel areas were calculated.Phase-contrast photographs were taken of the top and middle of each gelafter 1 and 2 days.

Results The changes in area observed for the collagen gels over the 2day test period are shown in FIG. 1. (In FIG. 1 mcg representsmicrograms.) It can be seen from the results that all the solutions ofcollagenase inhibitor inhibited the contraction of the collagen gels incomparison with the buffer solution (control 1) and the normal growthmedium solution (control 2). The amount of inhibition observed isconcentration dependent; the solution with the highest inhibitorconcentration showed the strongest inhibition of gel contraction. Thebuffer solution (control 1) also showed some inhibitory properties incomparison with the normal growth medium solution (control 2) but theeffect was not significant when compared to that exhibited by thebuffered collagenase inhibitor solution of even the lowestconcentration. At the end of the tests the cells in the collagen gelswere still alive and phase contrast microscopy showed that they appearedto be viable.

Discussion

It appears that MMPs are involved in fibroblast mediated contraction ofcollagen and that the use of an inhibitor can restrict contractionwithout killing the cells. Furthermore, the amount of inhibition is dosedependent.

Example 2

The toxicity of GM6001 (Galardin (trade name)) and a second collagenaseinhibitor GM1489 (a derivative of GM6001) to fibroblasts was tested atvarious concentrations.

The toxicity of the inhibitors was tested by measuring DNA synthesis bytritiated thymidine incorporation. The assay was adapted from aprocedure previously described, Woost, P. G. et al 1992.

Human ocular fibroblasts (Tendon's capsule fibroblasts) were cultured inTrimix (DMEM/F-10/M-199) cell culture medium (available from GIBCO/BRL)supplemented with 10% bovine calf serum and grown to confluency in T-75tissue culture flasks. (That is flasks having a surface area of 75 cm².)The cells were split and seeded into 24 well plates at a density of1×10⁴ /well in 1.0 ml of medium with 10% bovine calf serum. The cellswere incubated for 24-36 hours or until they reached 60-70% confluency.The medium was then changed to serum-free Trimix and the cells wereincubated for an additional 24 hours. The cells were then incubated inquadruplicate wells for 24 hours with 1.0 μCi/well ³ H-thymidine in thefollowing conditions: serum-free medium (Trimix), medium plus 10% serum,medium plus 10% serum and vehicle, alone or with the inhibitors. Thevehicle was a buffered acetate solution. The solutions comprising mediumwith 10% serum and vehicle, alone or with inhibitors, were prepared bythe method described in Example 1, section (c), above, using Trimixinstead of the HEPES buffered DMEM. The inhibitors, GM6001 and GM1489,were each tested at concentrations of 80.0, 8.0, and 0.8 μg/ml. At theend of the incubation period, the wells were washed 3 times with PBS andfixed with 12.5% TCA for 10 min followed by methanol for 10 min. Theplates were air dried and the cells solubilized in 1.0 ml of 0.2 N NaOHat 37° C. for 1 hour. Radioactivity was determined by liquidscintillation counting 900 ml of the solubilized cells. The experimentsshow that there is no significant toxicity at any of the concentrationstested. The results are shown in FIG. 2. In FIG. 2 SF1 and SF2 stand forserum-free medium, 10% for medium plus 10% serum, V1 and V2 for mediumplus 10% serum and vehicle and the other results are for the inhibitorsolutions (comprising also media plus 10% serum and vehicle).

FIG. 2 shows the thymidine incorporation of the ocular fibroblasts atdifferent concentrations of each collagenase inhibitor.

Discussion

It can be seen that there is no significant reduction in thymidineuptake even with the highest concentrations of the inhibitors. Thisindicates that the reduction in collagen contraction found when usingcollagenase inhibitors is not due to inhibition of cell proliferation.

In the following Examples methods and materials substantially asdescribed in Example 1 are used except where it is indicated to thecontrary.

Example 3

This experiment was designed to investigate the effect of the followingMMP inhibitors: Galardin, BB-94 and antibodies to MMPs, 1, 2 3 and 9, onocular fibroblast mediated collagen contraction.

The preparation of materials was as described in Example 1 above exceptwhere indicated. Test solutions were used in approximately 3 ml doses.For all contraction experiments cell morphology was monitored by phasecontrast microscopy and the growth medium was changed every 3 to 4 days.

a) Cell number dependence

Collagen gels were seeded with either 100,000 or 500,000 cells. Theywere exposed to inhibitor and control solutions comprising 40 μg/ml and4 μg/ml of Galardin or equimolar hydroxamic acid (control) at anequivalent to 40 μg/ml of Galardin (100 M) (made up as described inExample 1(c) above). The gels were monitored for 7 days.

For results, see FIGS. 3A and 3B.

FIG. 3A shows the results for the collagen lattices (gels) populatedwith 500,000 cells per lattice. The mean lattice area is plotted againsttime (in days) for gels treated with each of the three solutions. As maybe seen, the contraction of the gels exposed to inhibitor solutions ismuch less than that of gels exposed to the control solution. FIG. 3Bshows the results for the collagen lattices populated with 100,000 cellsper lattice. Again the mean lattice area is plotted against time foreach of the three regimes. As may be seen, the contraction of the gelsexposed to inhibitor solutions is much less than that of gels exposed tocontrol solutions. Comparing FIGS. 3A and 3B shows that the contractionof lattices populated with 500,000 cells per lattices is greater thanthat of lattices populated with 100,000 cells under the same conditions.

Discussion

The experiments confirmed that contraction is stronger when there is ahigher cell concentration and that the same dose of inhibitor cannotthen provide such good inhibition as when there is a lower cellconcentration.

b) Reversibility

Collagen gels were prepared and seeded with fibroblasts (1×10⁵cells/lattice (gel)) as described in Example 1 above. They were exposedto inhibitor and control solutions in the following ways:

(i) continual exposure to control solution (medium containing hydroxamicacid);

(ii) continual exposure to inhibitor solution (medium containing 4 μg/mlGalardin);

(iii) continual exposure to inhibitor solution (as (b)) until day 14post seeding and then replacement by control solution (as (a));

(iv) lattices were allowed to contract for 5 days and then werecontinually exposed to control solution (as (a)) for 25 days;

(v) lattices were allowed to contract for 5 days and then werecontinually exposed to inhibitor solution (as (b)) for 25 days;

(vi) lattices were allowed to contract for 5 days and then werecontinually exposed to inhibitor solution (medium containing 40 μg/mlGalardin) for 25 days.

The test solutions were made up in accordance with Example 1 (c) and thegrowth medium in the cultures was changed every 3 to 4 days.

The results of (i), (ii) and (iii) are shown in FIG. 3C and the resultsof (iv), (v) and (vi) are shown in FIG. 3D.

FIG. 3C is a plot of mean lattice area against time showing the resultsfor experiments (i), (ii) and (iii). As may be seen, the mean area oflattices treated with control solution decreases from day 0; contractionoccurs for the whole period. For the lattices initially treated withinhibitor solutions there is very little contraction and in the casewhere the lattices continue to be exposed to inhibitor (ii) thereduction in mean area is low even after 40 to 50 days. For the latticesexposed to inhibitor solution until day 14 (shown as * in FIG. 3C) andthereafter exposed to control solution (experiment (iii)) there is asignificant increase in contraction from day 14 onwards.

FIG. 3D is a plot of mean lattice area against time for each ofexperiments (iv), (v) and (vi). As may be seen, in the first 5 days ofthe experiments when lattices were not exposed to test solutions thelattices underwent substantial contraction. After day 5 (marked as * inFIG. 3D) the lattices were exposed to the test solutions. Thecontraction continued (there was no reversal) but at a reduced rate.Both the 4 μg/ml and 40 μg/ml solutions of Galardin inhibitedcontraction in comparison with the control solution. The 40 μg/mlsolution of Galardin gave a stronger inhibitory effect than the 4 μg/mlsolution.

Discussion

Experiments (i), (ii) and (iii) showed that exposure to inhibitorsolution inhibited contraction in comparison with exposure to thecontrol solution and that when the inhibitor solution was then replacedby control solution the rate of contraction increased, i.e. inhibitionstopped. Hence the inhibition is reversible.

Experiments (iv), (v) and (vi) showed that even when contraction of thegels had been allowed to take place for 5 days without any type ofinhibition, the addition of inhibitor solutions still gave inhibition ofcontraction. The more concentrated inhibitor solution gave greaterinhibition.

c) Cytotoxicity

This experiment was carried out substantially according to the method ofExample 2, except that cells were seeded at 2×10⁴ cells per well,cultured without radioactive material and were exposed to control medium(DMEM/10% NCS) or Galardin at 40 μg/ml and 4 μg/ml concentration.Approximately 1 ml of test solution was added in each case. Wells wereharvested and cells counted on a haemocytometer, see FIG. 3E for theresults, and the percentage of viable cells in each well was measuredusing the trypan blue dye exclusion test, see FIG. 3F for the results.As can be seen the Galardin did not adversely affect cell viability andnumber compared to controls.

Discussion

It appears that MMP inhibitors may be used in quantities andconcentrations high enough to restrict contraction without significanttoxicity.

d) BB-94

The experiment was carried out as for (a) above with 1×10⁵ cells perlattice and the test solutions used were BB-94 4 μg/ml, BB-94 0.4 μg/mland control with vehicle (0.1% vol/vol DMSO). The development of thelattices was followed for seven days after seeding. For the results seeFIG. 3G, from which it is clear that BB-94 gave significant inhibitionof contraction.

e) Antibodies

The experiment was carried out as for (d) above except that the testsolutions used were 1:50 (vol/vol) of antibodies in growth medium. Theantibodies used were antibodies to MMPs 1, 2, 3 and 9, obtained fromBiogenesis Ltd., (Poole, U.K.). A control solution of 0.1% sodium azideand 0.14% rabbit serum in PBS diluted to 1:50 in growth medium was alsoused. See FIG. 3H for the results.

FIG. 3H is a plot of mean lattice area against time for lattices exposedto each of the test solutions. As may be seen, the lattices exposed tocontrol solution and to the solution of antibody to MMP9 showedsignificant contraction. The lattices exposed to one of the antibodiesto MMP1, MMP2 or MMP3 showed no significant contraction.

Discussion

As may be seen from FIG. 3H significant inhibition was achieved withantibodies for MMPs 1, 2 and 3 but not with the antibody for MMP9. Thistherefore shows that antibodies may act as satisfactory MMP inhibitorsfor use in inhibiting contraction. It also appears that while MMPs 1, 2and 3 may all be involved in the contractile process MMP9 is not.

Example 4

A series of experiments were carried out in which the MMP mRNA andprotein production of human Tenon's capsule fibroblasts, under a numberof conditions, was analysed.

Fibroblast cells were both grown in monolayer culture and were seeded incollagen lattices. Total RNA was isolated from samples containing 3×10⁶cells using the method described in Chomczynski & Sacchi 1987. Sampleswere taken from cells in monolayer culture on day 0 of the experimentand samples were also taken from cells, which were contracting collagenlattices after 9 hours, 1 day and 7 days.

1 μ/mg samples of the RNA were treated with known copy numbers of asynthetic RNA template (0.1 to 100,000 copies) containing complementarysequences to the primers for sequences to MMPs 1, 2, 3 and 9. Themixtures underwent reverse transcriptase reactions prior toamplification by PCR using specific primers for MMPs 1, 2, 3 and 9.During the PCR reaction both the sample RNA and the synthetic templateRNA compete equally for primer binding.

Band intensities were image analysed and the ratio of amplifiedsynthetic template to amplified sample intensities calculated. Thesevalues were plotted versus initial template copy number. When the ratioof amplified synthetic template to sample is one, then the initial copynumbers of synthetic template and sample are equal. This allowedcalculation of message copy number per cell in all samples.

Samples of conditioned medium and collagen lattices (1×10⁵cells/lattice) were collected on days 1, 3 and 7 post seeding. Latticeswere washed in phosphate buffered saline (PBS; 3×3ml) prior tohomogenisation in 0.5% (vol/vol) Triton-X100 in PBS [Hunt et al. 1993].Gelatin zymography [Heussen & Dowdle 1980] was then performed on thesesamples. Briefly, samples and prestained molecular weight standards wereresolved on 10% tris glycine gels containing 0.1% gelatin (Novex, R&DSystems, Oxford, U.K.). Gels were then incubated for 30 minutes inrenaturing buffer, 30 minutes in developing buffer followed by a further18 hours in developing buffer at 37° C. (all Novex). Gels were thanstained in 0.5% (wt/vol) commassie blue in 45% (vol/vol) water, 45%(vol/vol) methanol and 10% (vol/vol) glacial acetic acid, followed bydestaining in 45% (vol/vol) water, 45% (vol/vol) water, 45% (vol/vol)methanol and 10% (vol/vol) glacial acetic acid. MMP activity appeared asclear bands on a blue background.

Results

The experiments showed that the RNA extracted from cells which werecontracting collagen contained raised levels of mRNA for MMPs 1, 2 and 3but not 9, in comparison with that taken from cells in monolayercultures. They also showed that levels of MMP mRNA decreased over the 7day culture period.

    ______________________________________                                                mRNA Copy Number/10.sup.6 Cells                                       MMP     Monolayer                                                                              9 Hours     1 Day 7 Days                                     ______________________________________                                        1       8        71          143   3                                          2       <1       15          144   81                                         3       <1       18          132   16                                         9       <1       <1          <1    <1                                         ______________________________________                                    

Also see FIG. 6 for results. FIG. 6 is a reproduction of a photograph ofthe results of the PCR reaction. It shows the sample and template bandsat day 0, 9 hours, 1 day and 7 days for mRNA encoding each of MMPs 1, 2,3 and 9.

The gelatin zymography showed that actively contracting cells whencompared to cells from monolayer cultures produced four proteolyticallyactive species (Mr approximately 100,000; 90,200; 72,000 and 57,000),two of which appear (ones with Mrs of 72,000 and 57,000) to increaseover the 7 day period. See FIG. 7 for results. One was activated uponincubation with aminophenyl mecuric acetate (Mr 100,000 reduced to57,000).

Discussion

The experiments showed that both MMP mRNA and protein production byocular fibroblasts is dramatically increased upon culture within, andduring the contraction of, a three dimensional collagen matrix. Ittherefore appears that active production of MMPs occurs during thecontractile process.

Further experiments showed that treatment of seeded lattices withGalardin resulted in the abolishment of the four proteolytically activespecies.

Example 5

This series of experiments investigated the effect of MMP inhibitors onthe degree of contraction of collagen lattices by fibroblasts fromvarious tissue sites and species.

Collagen lattices (gels) were prepared as previously described inExample 1 and were seeded with 1×10⁵ cells per lattice of:

i) human dermal fibroblasts;

ii) rat parietal sheath fibroblasts; or

iii) rat endotendon fibroblasts.

The lattices were then exposed to growth medium containing Galardin at40 μg/ml or hydroxamic acid at 100 μM (control). The test solutions weremade up as described in Example 3 above and approximately 3ml dose ofthe appropriate test solutions were used. The area of each lattice wasmeasured over 7 days and the results are shown in FIGS. 4A, 4B and 4C.

FIGS. 4A, 4B and 4C are plots of mean lattice area over time forlattices populated with fibroblasts under exposure to either controlsolution or inhibitor solution. FIG. 4A for human dermal fibroblasts, 4Bfor rat parietal sheath fibroblast and 4C for rat endotendon fibroblast.As may be seen, the contraction of lattices populated with any of thethree types of fibroblasts was greatly reduced when exposed to theinhibitor solutions in comparison with the lattices populated with thesame type of fibroblasts and exposed to the control solution.

Discussion

Exposure to the inhibitor resulted in a significant degree of inhibitionof contraction compared to controls in all of the cell types. In eachcase, inhibition of contraction was accompanied by a decreased cellularinvasion into and migration through, the surrounding matrix compared tocontrols. Therefore it appears that inhibition of cellular invasion isan important mechanism of MMP inhibitor action.

Example 6

The effect of an MMP inhibitor on the contraction of an artificial skinequivalent was tested. A collagen lattice (gel) was prepared as inExample 1 and was seeded with both human keratinocytes and human dermalfibroblasts to mimic skin. This lattice was then exposed to growthmedium containing Galardin at 40 μg/ml or hydroxamic acid at 100 μM(control). The solutions were made up as described in Example 3(a) aboveand approximately 3ml portions of the appropriate solutions were used.The lattice area was measured over 7 days and the results are shown inFIG. 5.

FIG. 5 is a plot of the mean lattice area over time (in days) forlattices exposed to the control solution and for lattices exposed to theinhibitor solution. As may be seen, the lattices exposed to theinhibitor solution suffered less contraction than those exposed to thecontrol solution.

Discussion

It is therefore possible to inhibit the contraction of a collagen gelwith fibroblasts and epithelial cells, i.e. an approximate model ofskin.

Example 7

The effects of MMP inhibitors on ocular fibroblast morphology withincollagen lattices were studied in this series of experiments.

Collagen lattices seeded with ocular fibroblasts were prepared andexposed to MMP inhibitors, both synthetic chemicals and antibodies, asdescribed in Example 3 above. Cellular morphology was monitored by phasecontrast microscopy.

Cells populating control collagen lattices exhibited stellate (S) andbipolar (B) morphology by 7 days post seeding. Small cytoplasmicprojections were exhibited by cells exposed to 4 μg/ml Galardin, 4 μg/mlBB-94, antibody to MMP1 or MMP2 at 7 days post seeding. Cells exposed toMMP3 antibody did not produce any cytoplasmic projections into thesurrounding matrix. Exposure to antibody to MMP9 did not affectmorphology compared to controls.

Discussion

These results showed that cells populating lattices exposed to Galardin,BB-94 and antibodies to MMPs 1, 2 and 3 exhibited decreased invasioninto the surrounding collagen matrix compared to controls and to cellsexposed to an antibody to MMP9. This decrease in invasion into thematrix was always accompanied by the inhibition of both latticecontraction and migration through the matrix. This again showed thatinhibition of invasion is important.

Example 8

This series of experiments was designed to investigate the effects of anMMP inhibitor (Galardin) on Tenons capsule fibroblast cellular processesrequired for collagen contraction.

Monolayer cultures of fibroblasts and collagen lattices (gels) seededwith 1×10⁵ cells per lattice were prepared (as described in Example 1).

The degree of contraction of collagen gels treated with control solutionand with inhibitor solution containing 40 μg/ml Galardin (solutions weremade up as described in Example 1 and approximately 3 ml portions ofsolutions were used) was monitored, as described above. FIG. 8A showsthe degree of lattice contraction of a control gel 7 days post seedingand FIG. 8B shows the degree of contraction of the gel which was exposedto inhibitor solution.

The actin cytoskeleton of cells in lattices and in monolayer culture wasimmunofluorescently stained with FITC-phalloidin [Martin & Lewis 1992].See FIGS. 9A (control) and 9B (exposed to inhibitor). The arrowsindicate the actin stress fibres on the surface of the cells populatingthe collagen gels. These fibres are clearly present in the control butdo not appear to be present in the lattice that was exposed to inhibitorsolution.

Differences between the controls and those exposed to inhibitor (40μg/ml Galardin) were seen. In order to assess whether this differencewas owing to the direct effect of the inhibitor or to differences in thedegree of invasion into the matrix, cells were exposed in monolayerculture to the inhibitor and the cytoskeleton was stained. See FIGS. 10A(control) and 10B (exposed to inhibitor). The arrows indicate the stressfibres on the cells in the monolayer culture. As may be seen, they arepresent in both the control monolayer culture and in the monolayerculture exposed to the inhibitor solution.

The inhibitor did not appear to affect the actin cytoskeleton comparedto controls in monolayer culture.

Lattices for transmission electron microscopy were harvested 3 days postseeding, washed with PBS (3×3ml), and fixed overnight at 4° C. in 2.5%(vol/vol) glutaraldehyde with 0.5% (wt/vol) tannic acid in 0.07M sodiumcacodylate buffer (pH 7.0). Following a rinse in 0.01M cacodylate-HClbuffer (pH 7.3), postfixation for 2 hours in 1% (wt/vol) aqueous osmiumtetroxide at 4° C., lattices were dehydrated through ascending alcoholsand cleared in propylene dioxide. Samples were then infiltrated withpropylene dioxide/araldite (1:1 vol/vol) for 1 hour, followed by 12hours immersion in Araldite (trade mark) alone. Samples were thenembedded in fresh Araldite, ultrathin sections cut and sequentiallystained with saturated uranyl acetate followed by Reynolds lead citrate.See FIGS. 11A (control) and 11B (exposed to inhibitor). The arrow headsindicate the cellular attachments to the collagen matrix. It is clearthat the attachment of the fibroblasts to their surrounding collagenmatrix was not affected by the inhibitor.

Discussion

These experiments showed that Galardin did not affect the cytoskeletonor cell matrix attachment of the fibroblasts. Effects on thecytoskeleton or on cell matrix attachment were possible explanations forthe effect of Galardin on contraction and therefore for the effect ofMMP inhibitors on contraction.

GENERAL DISCUSSION

A number of studies have suggested possible mechanisms for cell mediatedcollagen contraction. A number of workers have suggested that MMPs arenot produced or are produced during lattice contraction but are notimplicated in the contractile process. The above-described experimentsdemonstrate that cellular derived MMP activity is crucial to the processof collagen contraction. The requirement of MMP activity for contractionappears to be common to all of the fibroblasts tested.

The present invention is not to be limited in scope by the embodimentsdisclosed in the Examples which are intended to illustrate theinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art and are intended to fall within the scope of the appendedclaims.

BIBLIOGRAPHY

Eisen A. Z., Block K. L., Sakai T., Inhibition of human skin collagenaseby human serum, J. Lab. Clin. Med. 1976; 75: 258-263.

Vater C. A., Mainardi, C. L., Harris E. D. Jr, Native cross-links incollagen fibrils induce resistance to human synovial collagenase.Biochem. J. 1979; 181: 639-645.

Stricklin G. P., Welgus H. G., Bauer E. A., Human skin collagenase inrecessive dystrophic epidermolysis bullosa: Purification of a mutantenzyme from fibroblast cultures. J. Clin. Invest. 1983; 69: 1373-1383.

Murphy G., McGuire M. B., Russell R. G. G., Reynolds J. J.,Characterization of collagenase, other metalloproteinases and aninhibitor (IMP), produced by human synovium and cartilage in culture.Clinical Science 1981; 61: 711-716.

Welgus H. G., Kobayashi D. K., Jeffrey J. J., The collagen substratespecificity of rat uterus collagenase. J. Biol. Chem. 1983; 258:14162-14165.

Welgus H. G., Stricklin G. P., Human skin fibroblast collagenaseinhibitor: comparative studies in human connective tissues, serum andamniotic fluid. J. Biol. Chem. 1983; 258: 12259-12264.

Bar-Shavit Z., Teitelbaum S. L., Stricklin G. P., Eisen A. Z., Kahn A.J., Welgus H. G., Differentiation of human leukemia cell line andexpression of collagenase inhibitor. Proc. Nat. Acad. Sci. USA 1985; 82:5380-5384.

Wooley D. E., Roberts D. R., Evanson J. M., Small molecular weight serumprotein which specifically inhibits human collagenases. Nature 1976;261: 325-327.

Cooper T. W., Eisen A. Z., Stricklin G. P., Welgus H. G., Plateletderived collagenase inhibitor: characterisation and subcellularlocalisation. Proc. Natl. Acad. Sci. USA. 1985; 82: 2771-2783.

Grobelny D., Poncz L., Galardy R. E., Inhibition of human skinfibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastaseby peptide hydroxamic acids. Biochem. 1992; 31: 7152-7154.

Mullins D. E. etal, Biochem. Biophys Acta (1983) 695: 117-214.

Schultz G. S., Strelow S., Stern G. A., Chegini N., Grant M. B., GalardyR. E., Grobelny D., Rously J. J., Stonecipher K., Parmley V. and Khaw P.T. Treatment of Alkali-Injured Rabbit Corneas with a Synthetic Inhibitorof Matrix Metalloproteinases. Investigative Ophthalmology and VisualScience Vol 33, No. 12: 3325-3331.

Woost P. C., Jumblatt, M. M., Eiferman R. A., and Schultz G. S., GrowthFactors in Corneal Endothelial Cells: I. Stimulation of Bovine CornealEndothelial Cell DNA Synthesis by Defined Growth Factors. Cornea, 1992;11(1): 1-10.

Khaw P. T., Ward S., Porter A., Grierson I., Hitchings R. A., Rice N. S.C., The Long Term Effects of 5. Fluorouracil and Sodium Butyrate onHuman Tenons Capsule Fibroblasts. Investigative Ophthalmology and VisualScience 1992; 33: 2043-2052.

Nagase H., Barrett A. J., Woessner J. F. Jr., Nomenclature and Glossaryof Matrix Metalloproteinases. MATRIX (Suppl) 1992; 1:421-424.

Gabbiani G., Hirschel B. J., Ryan G. B., and et al. Granulation tissueas a contractile organ. A study of structure and function. J. Exp. Med.1972; 135:719-734.

Ehrlich H. P. and Rajaratnam J. B. M. Cell locomotion forces versus cellcontraction forces for collagen lattice contraction: an in vitro modelof wound healing. Tiss. Cell 1990; 22: 407-417.

Schor S. L., Allen T. D. and Harrison C. J. Cell migration throughthree-dimensional gels of native collagen fibres: collagenolyticactivity is not required for the migration of two permanent cell lines.J. Cell Sci. 1980; 46: 171-186.

Nakagawa S., Pawelek P. and Grinnell F. Long term culture of fibroblastsin contracted collagen gels: effects on cell growth and biosyntheticactivity. Journal of Investigative Dermatology 1989; 93: 792-798.

Maunch C., Adelmann-Grill B., Hatamochi A., and Krieg T. Collagenasegene expression in fibroblasts is regulated by a three-dimensionalcontact with collagen. FEBS. Letts. 1989; 250(2): 301-305.

Lambert C. A. Pretranslational regulation of extracellular matrixmacromolecules and collagenase expression in fibroblasts by mechanicalforces. Lab. Invest. 1992; 60(4): 444-451.

Tarnuzzer R. W. and Schultz G. S. Quantitative competitive RT-PCRtechnique for growth factors and their receptors: applications in thestudy of corneal wound healing. Invest. Ophthalmol. Vis. Sci. 1994;35:1318.

Heussen C. and Dowdle E. B. Electrophoretic analysis of plasminogenactivators in polyacrylamide gels containing sodium dodecyl sulfate andcopolymerized substrates. Analyt. Biochem. 1980; 102: 196-202.

Chomczynski P. and Sacchi N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem.1987; 162: 156-159.

Hunt R. C., Fox A., Pakalnis V. A., Sigel M. M., Kosnosky W., ChoudhuryP., and Black E. P. Cytokines cause cultured retinal pigment epithelialcells to secrete metalloproteinases and to contract collagen gels.Invest. Opthalmol. Vis. Sci. 1993; 34: 3179-3186.

Martin P. and Lewis J. Actin cables and epidermal movement in embryonicwound healing. Nature 1992; 360: 179-182.

We claim:
 1. A method for inhibiting or preventing contraction ofhealing tissue following trauma selected from the group consisting ofsurgical or accidental cut or tear, in which matrix metalloproteinaselevels are not substantially elevated, or of cultured tissue, saidtissue comprising an extracellular matrix component, said methodcomprising the step of administering an effective amount of a matrixmetalloproteinase inhibitor to said tissue such that said contraction isinhibited or prevented.
 2. The method of claim 1 wherein said tissue istissue of the eye.
 3. The method of claim 1 wherein said matrixmetalloproteinase inhibitor is administered in a concentration ofgreater than 400 μg/mL.
 4. The method of claim 1 wherein said tissue iscultured tissue.
 5. The method of claim 4 herein said tissue is skingraft tissue.
 6. The method of claim 4 wherein said tissue is tissue ofthe eye.
 7. The method of claim 4 wherein said matrix metalloproteinaseinhibitor is administered at a concentration at least about 0.4 μg/mLand less than 40 μg/mL.
 8. The method of claim 4 wherein said matrixmetalloproteinase inhibitor is administered at a concentration more thanabout 400 μg/m.
 9. The method of claim 1 wherein said matrixmetalloproteinase inhibitor is a collagenase inhibitor.
 10. The methodof claim 1 wherein said matrix metalloproteinase inhibitor is capable ofinhibiting a matrix metalloproteinase selected from the group consistingof matrix metalloproteinases 1, 2 and
 3. 11. The method of claim 1wherein said matrix metalloproteinase inhibitor isN-[2(R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]L-tryptophanmethylamide.
 12. The method of claim 1 wherein said matrixmetalloproteinase inhibitor is a peptide hydroxamic acid or apharmaceutically acceptable derivative thereof.
 13. The method of claim1 wherein said matrix metalloproteinase inhibitor is a monoclonal orpolyclonal antibody to a matrix metalloproteinase.
 14. The method ofclaim 1 wherein said matrix metalloproteinase inhibitor is a collagenaseinhibitor administered in combination with at least one other matrixmetalloproteinase inhibitor.
 15. The method of claim 1 wherein saidmatrix metalloproteinase inhibitor is administered in combination with afurther pharmacologically active ingredient selected from the groupconsisting of antibiotics, antifungals, steroids, enzyme inhibitors,epidermal growth factors, fibronectin and aprotinin.
 16. The method ofclaim 1 wherein said matrix metalloproteinase inhibitor is administeredin combination with an inhibitor of a collagen-stimulating cytokine. 17.A method of treating a human or other mammal to inhibit or preventcontraction of a post-trauma condition of a tissue selected from thegroup consisting of surgical or accidental cut or tear, in which matrixmetalloproteinase is not substantially elevated, said tissue comprisingan extracellular matrix component, said contraction being associatedwith healing of said tissue, comprising administering to said mammal aneffective amount of a matrix metalloproteinase inhibitor to inhibit orprevent such contraction at a concentration greater than 400 μg/mL. 18.The method of claim 17 wherein said contraction would occur as theresult of surgical treatment of the eye.
 19. The method of claim 17wherein said contraction would occur as the result of glaucoma surgery.